At present, the detection of pathogenic bacteria transmissible by food, such as Listeria spp, Staphylococcus aureus, Campylobacter jejuni and Escherichia coli O157:H7, is a very important task in the field of medicine and public health and is very important in the agro-foodstuffs, both for the producer and the distributor of food products (raw materials and/or processed products), for which several methods have been described for their detection and identification.
One of the current methodologies, considered among the most effective in the detection, identification and quantification of pathogens, is the one that is based on molecular techniques, as the method of polymerase chain reaction, commonly known as PCR. The PCR procedure is generally regarded as the most sensitive and rapid method used to detect nucleic acids of pathogens in a given sample test in particular, and we can find it described within the state of the art by Kary B. Mullis et al. in the family of U.S. Pat. No. 4,683,195, U.S. Pat. No. 4,683,202, U.S. Pat. No. 4,800,159, U.S. Pat. No. 4,889,818, U.S. Pat. No. 4,965,188, U.S. Pat. No. 5,008,182, U.S. Pat. No. 5,038,852, U.S. Pat. No. 5,079,352, U.S. Pat. No. 5,176,995, U.S. Pat. No. 5,310,652, U.S. Pat. No. 5,310,893, U.S. Pat. No. 5,322,770, U.S. Pat. No. 5,333,675, U.S. Pat. No. 5,352,600, U.S. Pat. No. 5,374,553, U.S. Pat. No. 5,386,022, U.S. Pat. No. 5,405,774, U.S. Pat. No. 5,407,800, U.S. Pat. No. 5,418,149, U.S. Pat. No. 5,420,029 among others.
To perform the PCR technique, basically, you need at least one pair of oligonucleotides for each of the pathogens to be identified, so that each pair of primers include a first nucleotide sequence complementary to a sequence that borders on the extreme 5′ of a sequence of a nucleic acid target and a second nucleotide sequence complementary to a sequence that borders the 3′ end of the sequence of nucleic acid target. Nucleotide sequences should have every pair of oligonucleotide primers which are specific to the pathogen to be detected, so that they do not react or are crossed with other pathogens.
As the PCR technique is a sensitive and quick method to detect pathogens on an individual basis, this can also be used to simultaneously detect multiple pathogens present in a sample. However, the PCR methodology used for simultaneous detection of multiple pathogens in a sample is problematic, since its main obstacle lies in the cross reaction that may be present due to the use of multiple nucleotide sequences in order to have the preferential amplification of certain target sequences, present in the sample at the expense of other target sequences, also present.
Examples of multiple and simultaneous detection of pathogens, using the PCR methodology, are described by John W. Czajka in the issuance of international patent application WO-0314704, and by Linxian Wo and others in the family of U.S. Pat. No. 5,612,473, U.S. Pat. No. 5,738,995, U.S. Pat. No. 5,753,444, U.S. Pat. No. 5,756,701, and U.S. Pat. No. 5,846,783.
The publication of international patent application WO-0314704 describes a method to detect specific and simultaneously pathogen species of Campylobacter in a complex test sample. The pathogen species of Campylobacter to be detected may be Campylobacter jejuni or Campylobacter coli. The complex test sample can be a sample of food, water or a rich array of food. The method uses the PCR amplification with or without an internal positive control and appropriate pairs of primers. Multiple species can be detected in said reaction.
In the family of U.S. Pat. No. 5,612,473, U.S. Pat. No. 5,738,995, U.S. Pat. Nos. 5,753,444, 5,756,701, and U.S. Pat. No. 5,846,783 a multiplex PCR method is described to detect infectious agents quickly and simultaneously in one sample. The infectious agents that are detected, are Salmonella spp, Shigella spp, Campylobacter spp, Yersinia spp and Escherichia coli, in particular Escherichia coli O157:H7. The limitation of the method described in these patents is that it allows a minimum cross-reaction between the oligonucleotides and probes, as well as of the first ones with other sequences of nucleic acid during the amplification.
Other molecular methods that are currently used, there are some on the market to detect pathogens in food, some through DNA hybridization (Gene-Trak systems, Unipath), which is very sensitive but requires about 50 hours, and others by means of nucleic acid amplification (BAX, Dupont and FOMS Probelia, Sanofi Diagnostic Pasteur) requiring at least 24 hours. None of these methods provides results on the same day of the production of food, nor makes any quantification of the present pathogenic contamination.
Among the methods described above, in some cases it will be necessary only to increase the sensitivity of the method to detect the presence or absence of pathogens in particular reliably and rapidly, whereas in other cases it may be necessary, moreover, to further quantify pathogens that may present in order to establish the limits of concentration, starting from which the presence of the pathogen may present a problem for the consumer's health.
According to the former, it is of utmost concern to the food and health industry to have a quick method, which employs less than 5 hours, to detect and quantify simultaneously four of the most important infectious agents or pathogens, transmissible by food and/or contaminated environmental surfaces, such as Listeria spp, Staphylococcus aureus, Campylobacter jejuni, and Escherichia coli O157:H7. This detection and multiple, simultaneous and quick quantification of pathogens, through reaction of multiplex amplification using polymerase chain reaction in real time, will allow to save costs and time in an industry where the times on the shelf of the products are extremely important.